Surface display technology, where a protein, such as peptide and polypeptide, is attached and expressed on the surface of organism, can be applied to a variety of bioengineering fields according to the properties of the expressed protein or, the properties of host cells, on the surface of which the protein will be expressed (Georgiou et al., TIBTECH, 11:6-10, 1993; Nature Biotechnol., 15:29-34, 1997; and Lee et al., TIBTECH, 21:45-52, 2003). This surface display technology was developed using various cellular organisms, such as bacteriophages, bacteria, yeasts or mammal cells, as host cells.
In order to express a foreign protein on the surface of cells using the outer membrane protein of a certain organism, a suitable surface protein and the foreign protein should be linked with each other at a gene level to biosynthesize a fusion protein, and stably passed through a cell inner membrane and attached, and then maintained on the cell surface. For this purpose, a protein having the following properties is preferably selected for use as a matrix for surface expression. Namely, (1) it has a secretion signal capable of passing through the cell inner membrane, at the N-terminal end; (2) it must have a targeting signal which can be stably attached on a cell outer membrane; (3) it can be expressed on the cell surface in large amounts within the range having no adverse effect on the growth of cells, such that the protein can show high activity; and (4) it must be able to be stably expressed regardless of its size such that it can be used in various reactions (Georgiou et al., TIBTECH, 11:6-10, 1993). Such a matrix for surface expression needs to be genetically engineered such that it is inserted into the N-terminal end, C-terminal end or central portion of the outer membrane protein on the surface of the host cells (Lee et al., TIBTECH, 21:45-52, 2003).
Phage surface display technology became very important, since it is advantageous in most rapidly obtaining monoclonal mutants from a large amount of a library (106-1012 mutants) and thus applied to the ultra-high speed screening of antibodies. However, in the phage surface display technology, the level of surface expression of the screened antibodies from a library expressed on the surface of phages was very low. Also, in a surface expression system for gram-negative bacteria, the insertion of a foreign polypeptide resulted in a structural limitation such that stable membrane proteins could not be formed (Charbit et al., J. Immunol., 139:1644-58, 1987; and Agterberg et al., Gene, 88:37-45, 1990). Also, the stability and viability of the surface of host cells were reduced. E. coli hosts, on which the surface expression technology have been most intensively studied, was developed using a cell surface protein as matrix for surface expression, but had a disadvantage in that if the cell surface protein is over-expressed in a fused form with a foreign protein, the cell surface becomes structurally unstable and thus the viability of the host cells is reduced (Georgiou et al., Protein Eng., 9:239-47, 1996).
Meanwhile, spores are formed in vegetative cells during the growth terminating stage to break the vegetative cells, and then be exposed extracellularly. These spores are structurally uniform, stable, and have resistance to adverse effects of surroundings, such as ultraviolet rays, radiations, heat, toxic compounds and solvents, and thus can be used in various fields. Furthermore, when the environment suitable for cell growth is reached, the spores are germinated again and continued to grow to make it easy to amplify and collect.
There are a large number of cases where a spore coat protein is used as an expression matrix to express target proteins on the spore surface of microorganisms. Examples of such cases include: a case where β-galactosidase for which lacZ as a reporter gene codes is expressed on the surface of spore in a fused form with the outer shell coat protein (CotC) or inner shell coat protein (CotD) of Bacillus subtilis (U.S. Pat. No. 5,766,914; U.S. Pat. No. 5,837,500; and U.S. Pat. No. 5,800,821); a case where bacteria and viral/pathogenic antigens are surface-expressed using coat proteins, including CotC, CotD, CotA, CotB, CotE, CotF, CotG, CotN, CotS, CotT, CotV, CotW, CotX, CotY, and CotZ, as a matrix for spore surface display (US 2002/0150594 A1); and a case where target proteins are expressed on the surface of Bacillus subtilis spores, using coat proteins, including CotE, CotG, CotA, CotB, CotC, CotD, CotF, CotH, CotJA, CotJC, CotK, CotL, CotM, CotS, CotT, CotV, CotW, CotX, CotY, CotZ, SpoIVA, SpoVID, and SodA, as a matrix for spore surface display (WO 02/46388).
However, if the spore coat protein is used as the expression matrix, a fusion protein with the surface expression matrix should be made for the surface expression of target proteins, but there is a disadvantage in that the expression level of the fusion protein is low since one of various coat proteins is used as a matrix for surface expression. Also, the level of the surface expression of the target protein using the surface expression matrix and the fusion protein is limited since the target protein is expressed depending on a degree at which the surface expression matrix is inserted into the surface of cells and spores. Furthermore, if the target protein is over-expressed, there are problems in that the structural deformation of the spores can be caused or the inherent physical and chemical properties of the spores can be greatly changed, so that the viability or resistance to environment of the host cells can be greatly reduced.
Accordingly, the present inventors have conducted intensive studies in an attempt to solve the problems occurring in the above-mentioned spore surface display technology of the prior art, and consequently found that if a target protein was expressed on the cell or spore surface using the structurally stable exosporium of Bacillus subtilis as a matrix for surface expression, the target protein fused with the exosporium can be surface-expressed without changing the inherent structure of cells or spores even when the target protein is over-expressed, so that the viability or resistance to environment of the gene carriers (cells or spores) is not changed, thereby perfecting the present invention.
Since the exosporium is present on the outermost surface of a spore, it can show the effect of surrounding the whole spore by a target protein upon spore surface display without influencing the structure of the coat protein of a spore. Moreover, it can also be used in the cell surface expression of bacteria, including gram-positive or gram-negative bacteria using an exosporium protein. It has not yet been reported that a target protein can be expressed on the surface of cells or spores using the exosporium of the Bacillus cereus group was not yet reported. The Bacillus cereus group, which generally includes Bacillus thuringiensis and Bacillus anthracis, possesses a loose balloon-like exosporium, which has also been found in some other bacilli and clostridia (Desrosier, J. P. and J. C. Lara, J. Gen. Microbiol., 130:935-40, 1984).
If this exosporium is used in the surface expression of a target protein, the target protein will not influence the spore coat protein and thus will not cause the structural problem. Also, the amount of the target protein expressed on the spore surface is increased, and the viability or resistance to environment of a host cell will not be changed even when the amount of the target protein expressed on the outer membrane of the spores is increased.